Microtubule-based transport is vital for cell function. Proper transport of intracellular cargo involves an interplay between motor proteins that move at different speeds and in different directions, and microtubules, which can be oriented in either uniform or mixed polarity bundles and can also be altered by posttranslational modifications (PTMs) and microtubule associated proteins (MAPs). The dominant model in the field to describe the bidirectional motion is the tug-of-war model in which opposite-directed kinesin and dynein motors mechanically compete for dominance and control movement for short durations. However, a number of studies carried out in isolated cells and model organisms over the last 15 years have found that inhibiting one motor diminishes motility in both directions, just the opposite of the tug-of-war model prediction. The purpose of this proposal is to use mathematical modeling and in vitro experiments to investigate the mechanisms that cause the predictions of the tug-o-war model to fall short at the cellular scale. Closing this knowledge gap requires the integrated development of experiments and theoretical models in order to link fundamental mechanisms at the molecular scale with the observed within-cell transport behavior. The modeling will span three levels. Nanoscale modeling will use established frameworks to treat the kinetics of kinesin and dynein stepping and changes in motor-microtubule interactions due to tubulin PTMs and MAP binding. At the mesoscale, multiple motors bound to a common cargo are represented as continuous movers obeying stochastic differential models with performance characteristics taken from existing experimental data. Finally, at the microscale, the interaction of motor-cargo complexes with multiple microtubules and the dependence of transport on microtubule organization at the cellular level are treated. In Aim 1, in vitro and in silico experiments with defined numbers of kinesins and dyneins will be used to challenge specific hypothetical models of bidirectional transport. In Aim 2, this integrated approach will be extended to understand transport dynamics in cell-mimicking microtubule architectures in vitro and in silico.